Whey protein micelles

ABSTRACT

The present invention relates to whey protein micelles, particularly to whey protein micelle concentrates or powders thereof and to a method for producing them. The present invention also pertains to the use of these micelles concentrates or powders thereof in nutrition and/or cosmetics and/or pharmaceutics.

FIELD OF THE INVENTION

The present invention relates to whey protein micelles, particularly towhey protein micelle concentrates and powders thereof and to a methodfor producing them. The present invention also pertains to the use ofthese micelles concentrates and powders thereof in a wide range ofapplications.

BACKGROUND

Protein constitutes an indispensable part of the diets of many people.It is not only used for its nutritional value but also imparts desirabletexture and stabilisation to foods. For instance, in fat-containingproducts, the fat must remain stabilized over the entire shelf life ofthe product, so that no phase separation occurs.

To this end, emulsifying agents are utilised, that provide astabilization of the emulsion once formed, based on their inherentproperty of a lipophilic or hydrophobic part being soluble in thenon-aqueous phase and a polar or hydrophilic part being soluble in watersuch that said molecules facilitate emulsifying one phase in the otherphase. Additionally, the emulsifying agents also protect the once formeddroplets from aggregation and coalescence. As emulsifying agentsnaturally occurring substances are used, such as hydrocolloids,phospholipids (lecithin) or glycolipids and on the other hand syntheticagents like stearyl-2-lactylate or mono-, diacylglycerides, etc. mayalso be used.

One of the major drawbacks of the agents resides in that they sometimessubstantially add to the costs of the final product, and do not add tothe nutritional value of the product. Sometimes, such kinds of materialsalso do not show adequate stabilising properties because of aninterfacial competition with proteins.

Increasingly, therefore, protein is also being used as an emulsifier andas a partial substitute for fat.

U.S. Pat. No. 6,767,575 B1 discloses a preparation of an aggregate wheyprotein product, whereby whey protein is denatured by acidification andheating. The protein aggregates thus obtained are used in foodapplication.

GB 1079604 describes improvements in the manufacture of cheese, wherebywhey proteins undergo heat treatment at an optimum pH value, in order toobtain insoluble whey proteins which are then added to raw milk.

WO 93/07761 is concerned with the provision of a dry microparticulatedprotein product which can be used as a fat substitute.

U.S. Pat. No. 5,750,183 discloses a process for producing proteinaceousmicroparticles which are useful as fat substitute containing no fat.

A proteinaceous fat substitute is also disclosed in WO 91/17665 wherebythe proteins are in the form of a water-dispersible microparticulateddenatured whey protein.

Apart from the food applications, proteins are also present in manypharmaceutical and cosmetic compositions.

One of the problems encountered with the production of productscontaining globular proteins in general, and whey protein in particular,however is their limited processability in industrial food production.Indeed, protein molecules when heated, or when subjected to acidic oralkaline environment or in the presence of salts tend to lose theirnative structure and reassemble in various random structures such asgels, for example.

The preparation of gelled aqueous compositions of whey proteins is thesubject of EP 1281322.

Elofsson et al. in International Dairy Journal, 1997, p. 601-608describe cold gelling of whey protein concentrates.

Similarly, Kilara et al. in Journal of Agriculture and Food 20Chemistry, 1998, p. 1830-1835 describes the effect of pH on theaggregation of whey proteins and their gelation.

This gel effect presents limitation in terms of not only processability(e.g. clogging of machines used in the manufacture of protein-containingproducts) but also in terms of the texture thus obtained, which may notbe desirable for the wide range of protein applications.

Controlled denaturation of proteins is thus desirable in order to widenthe use of proteins.

In the Proceedings of the Second International Whey Conference, Chicago,October 1997, reported in International Dairy Federation, 1998, 189-196,Britten M. discusses heat treatments to improve functional properties ofwhey proteins. A process for producing whey protein micro-particledispersion at 95° C. is described.

Erdman in Journal of American College of Nutrition, 1990, p. 398-409describes that the quality of microparticulated protein is not affecteddespite using high shear and heat.

EP 0603981 also describes a heat stable oil-in-water emulsion containingproteins.

Sato et al. in U.S. Pat. No. 5,882,705 obtained micellar whey protein byheat treating a hydrolysed whey protein solution. The micellar wheyprotein are characterised by an irregular shape.

Thus, an object of the invention is to improve the usability of proteinsin industrial production processes.

SUMMARY OF THE INVENTION

Accordingly, this object is achieved by means of the features of theindependent claims. The dependent claims develop further the centralidea of the present invention.

To achieve this object, a method for the production of whey proteinsmicelles concentrates is proposed, in a first aspect, which comprisesthe steps of subjecting a solution containing native whey proteins to aspecific temperature at a specific pH and concentrating the solutionthus obtained to result in the production of a whey protein micelleconcentrate comprising whey protein micelles having a diameter of lessthan 1 μm.

In particular, the present invention relates to a process for theproduction of whey protein micelles concentrate comprising the steps of:

-   -   a. Adjusting the pH of a whey protein aqueous solution to a        value between 3.0 and 8.0,    -   b. Subjecting the aqueous solution to a temperature between 70        and below 95° C. and    -   c. Concentrating the dispersion obtained in step b.

In a second aspect, the invention relates to the whey protein micellesconcentrate thus obtainable and to whey protein micelles having aprotein concentration greater than 12%. In a further aspect, the presentinvention relates to the use of said concentrate in nutritional and/orcosmetic and/or pharmaceutical applications. A composition containingthe whey protein concentrate also falls under an aspect of the presentinvention.

Furthermore, the whey protein micelles concentrate may be dried, inparticular by freeze-drying, roller drying or spray-drying, yielding awhey protein micelles powder.

Thus, according to another aspect, the invention provides a whey proteinmicelles powder comprising at least 20% micelles.

The whey protein micelles concentrate may be spray-dried with additionalingredients thus resulting in a mixed whey protein powder comprisingwhey protein micelles and additional ingredients in a weight ratio of30:1 to 1:1000 according to a further aspect of the invention.

The use of the whey protein powder or the mixed whey protein powder forinstance in the production of protein enriched consumables andcompositions comprising these powders and, for instance, are allfeatures of the present invention.

Whey protein micelles and consumable products comprising said micellesare also features of the present invention.

FIGURES

The present invention will be further described hereinafter withreference to some preferred embodiments shown in the accompanyingfigures in which:

FIG. 1 shows the result of an experiment demonstrating the effect of pHand heat treatment on the micellisation of β-lactoglobulin.

FIG. 2 is showing a mean to determine the pH of micellisation for acommercial preparation (Bipro®, Batch JE032-1-420) using turbiditymeasurements at 500 nm.

FIG. 3 is a TEM (Transmission Electron Microscopy) micrograph from wheyprotein micelles (2 wt. %, WPI 95, Lactalis) at pH 7.4. Scale bar is 200nm.

FIG. 4 shows the result of an experiment evaluating the impact of theionic strength (Arginine HCl) on the formation of protein micelles atconstant pH of 7.0.

FIG. 5 shows the volume stability (FVS) of foam stabilized by 1 wt. %β-lactoglobulin micelles (Davisco) at pH 7.0 in presence of 60 mMArginine HCl compared to non-micellised β-lactoglobulin.

FIG. 6 shows the intensity-based equivalent hydrodynamic diameter ofwhey protein obtained by heat-treatment of a 1 wt % β-lactoglobulindispersion for 15 min at 85° C. at pH ranging from 2 to 8. Whey proteinmicelles are obtained at pH 4.25 (positively charged with a zetapotential around +25 mV) and at pH 6.0 (negatively charged with a zetapotential around −30 mV). Z-averaged hydrodynamic diameter of themicelles was 229.3 nm at pH 4.25 and 227.2 nm at pH 6.0. Thecorresponding micrographs of the micelles obtained by TEM after negativestaining are shown. Scale bars are 1 μm.

FIG. 7 shows a highly schematic structure of a whey protein micelle.

FIG. 8 shows a SEM (Scanning electron microscopy) micrograph of a wheyprotein micelle powder obtained after spray drying of a 20% proteincontent dispersion after microfiltration.

FIG. 9 is a negative staining TEM micrograph of a whey protein micellesdispersion obtained at 4% protein content.

FIG. 10 is a negative staining TEM micrograph of a whey protein micelledispersion obtained at 20% protein content after microfiltration.

FIG. 11 shows the heat stability of a whey protein micelle dispersionobtained at 10% protein content after microfiltration at pH 7.0 inpresence of NaCl after heating at 85° C. for 15 min.

FIG. 12 shows the heat stability of a whey protein dispersion obtainedat 4% protein content at pH 7.0 in presence of NaCl after heating at 85°C. for 15 min.

FIG. 13 is a negative staining TEM micrograph from a 4% whey proteinmicelles dispersion based on a pure whey protein micelle spray driedpowder after dispersion at 50° C. in deionised water.

FIG. 14 is a graph showing the size distribution of micelles obtained bythe process of the invention using a 4% Prolacta 90 whey protein isolatetreated at pH 5.9.

FIG. 15 is a SEM micrograph showing the internal structure after cuttingof a spray-dried powder granule that is presented on FIG. 8.

FIG. 16 is a negative staining TEM micrograph of a 4% whey proteinmicelles dispersion based on a pure freeze dried whey protein micellepowder after at room temperature in deionised water. Scale bar is 0.5micrometer.

FIG. 17 is a schematic view of the WPM coating by SBO (sulphated butyloleate) upon increasing the mixing ratio at pH 3.0. Grey circle: WPMwith positive surface charges. Black head+tail: negatively charged headand hydrophobic tail from SBO.

FIG. 18 is a photograph of a whey protein micelle concentrate at 20%obtained after evaporation in which 4% NaCl is added.

FIG. 19 is a bright field light microscopy micrograph of whey proteinmicelle powder semi-thin section after toluidine blue staining. Scalebar is 50 microns.

FIG. 20 is a SEM micrograph of the hollow whey protein micelle powderparticle after cutting. Left: internal structure. Right: Detail of thewhey protein micelle composing the powder particle matrix. Scale barsare 10 and 1 micron respectively.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 7 is a schematic representation of the micelles of the presentinvention, wherein the whey proteins are arranged in such a way that thehydrophilic parts of the proteins are oriented towards the outer part ofthe agglomerate and the hydrophobic parts of the proteins are orientedtowards the inner “core” of the micelle. This energetically favourableconfiguration offers good stability to these structures in a hydrophilicenvironment.

The specific micelle structure can be seen from the figures, inparticular FIGS. 3, 9, 10, 13 and 15, wherein the micelles of thepresent invention consist essentially of spherical agglomerates ofdenatured whey protein. The micelles of the present invention areparticularly characterised by their regular, spherical shape.

Due to their dual character (hydrophilic and hydrophobic), thisdenatured state of the protein seems to allow interaction with ahydrophobic phase, e.g. a fat droplet or air, and a hydrophilic phase.The whey protein micelles therefore have perfect emulsifying and foamingproperties. Furthermore, the micelles produced by the method of thepresent invention have an extremely sharp size distribution (see FIG.14), such that more than 80% of the micelles produced will have a sizesmaller than 1 micron, preferably between 100 nm and 900 nm, morepreferably between 100-770 nm, most preferably between 200 and 400 nm.

The mean diameter of the micelles can be determined using TransmissionElectron Microscopy (TEM). In order to do so, the liquid micelle samplesare encapsulated in agar gel tubes. Fixation is achieved by immersion ina solution of 2.5% glutaraldehyde in 0.1M, pH 7.4 cacodylate buffer andpost-fixation with 2% Osmium tetroxide in the same buffer, bothsolutions containing 0.04% Ruthenium red. After dehydration in a gradedethanol series (70, 80, 90, 96, 100% ethanol), the samples are embeddedin Spurr resin (Spurr/ethanol 1:1, 2:1, 100%). After polymerization ofthe resin (70° C., 48 hours), semi-thin and ultra-thin sections are cutwith a Leica ultracut UCT ultra-microtome. Ultra-thin sections, stainedwith aqueous uranyl-acetate and lead citrate, are then examined bytransmission electron microscopy (Philips CM12, 80 kV).

Without wishing to be bound by theory, it is thought that during micelleformation according to the process of the invention, the micelle reach a“maximum” size, due to the overall electrostatic charge of the micellerepelling any additional protein molecule, such that the micelle cannotgrow in size any longer. This accounts for the narrow size distributionobserved (cf. FIG. 14).

The micelles described above are produced by a process according to thepresent invention, said process being described in detail in thefollowing.

As the whey protein to be used in the present method, any commerciallyavailable whey protein isolates or concentrates may be used, i.e. wheyprotein obtained by any process for the preparation of whey proteinknown in the art, as well as whey protein fractions prepared therefromor proteins such as β-lactoglobulin (BLG), α-lactalbumin and serumalbumin. In particular, sweet whey obtained as a by-product in cheesemanufacture, acid whey obtained as a by-product in acid caseinmanufacture, native whey obtained by milk microfiltration or rennet wheyobtained as a by-product in rennet casein manufacture may be used as thewhey protein. The whey protein may be from a single source or frommixtures of any sources. It is preferable that the whey protein does notundergo any hydrolysis step prior to micelle formation. Thus, the wheyprotein is not subjected to any enzymatic treatment prior tomicellisation. According to the invention, it is important that the wheyprotein be used in the micelle formation process and not hydrolysatesthereof.

The present invention is not restricted to whey isolates from bovineorigin, but pertains to whey isolates from all mammalian animal species,such as from sheep, goats, horses, and camels. Also, the processaccording to the present invention applies to mineralised, demineralisedor slightly mineralised whey preparations. By “slightly mineralised” ismeant any whey preparation after elimination of free minerals which aredialyzable or diafiltrable, but which maintains minerals associated toit by natural mineralisation after preparation of the whey proteinconcentrate or isolate, for example. These “slightly mineralised” wheypreparations have had no specific mineral enrichment.

Whey proteins are an excellent source of essential amino acids (AA)(45%). Compared to casein (containing 0.3 g cysteine/100 g protein),sweet whey proteins contain 7 times more cysteine, and acid whey 10times more cysteine. Cysteine is the rate limiting amino acid forglutathione (GSH) synthesis, a tripeptide made of glutamate cysteine andglycine which has primary important functions in the defence of the bodyin case of stress. Requirements in these amino acids may be increased incase of stress and in elderly people. Also, glutathione oralsupplementation with whey protein has been shown to increase plasma GSHlevels of HIV-infected patients (Eur. J. Clin. Invest. 2001; 31,171-178).

Other health benefits provided by whey proteins include enhancement ofmuscle development and building, as well as muscle maintenance inchildren, adults or elderly people, enhancement of the immune function,improvement of cognitive function, control of blood glucose such thatthey are suitable for diabetics, weight management and satiety,anti-inflammatory effects, wound healing and skin repair, lowering ofthe blood pressure, etc.

Whey proteins have a better protein efficiency ratio (PER=118) comparedfor example to casein (PER=100). PER is a measure of a protein qualityassessed by determining how well such protein supports weight gain. Itcan be calculated by the following formula:PER=body weight growth(g)/protein weight intake(g).

Examples: PER % Casein casein 3.2 100 Egg 3.8 118 Whey 3.8 118 WholeSoya 2.5 78 Wheat gluten 0.3 9

For the process of the invention, whey proteins may be present in anaqueous solution in an amount of 0.1 wt. % to 12 wt. %, preferably in anamount of 0.1 wt. % to 8 wt. %, more preferably in an amount of 0.2 wt.% to 7 wt. %, even more preferably in an amount of 0.5 wt. % to 6 wt. %,most preferably in an amount of 1 wt. % to 4 wt. % on the basis of thetotal weight of the solution.

The aqueous solution of the whey protein preparation as present beforethe micellisation step may also comprise additional compounds, such asby-products of the respective whey production processes, other proteins,gums or carbohydrates. The solution may also contain other foodingredients (fat, carbohydrates, plant extracts, etc). The amount ofsuch additional compounds preferably does not exceed 50 wt. %,preferably 20 wt. %, and more preferably does not exceed 10 wt. % of thetotal weight of the solution.

The whey protein may be used in purified form or likewise in form of acrude product. According to a preferred embodiment, the content ofdivalent cations in the whey protein for the preparation of the wheyprotein micelles concentrate may be less than 2.5%, more preferably lessthan 2%, even more preferably less than 0.2%. Most preferably the wheyproteins are completely demineralised.

According to the present finding, the pH and the ionic strength areimportant factors in the present method. Thus, for extensively dialyzedsamples which are virtually devoid or depleted of free cations such asCa, K, Na, Mg, it has been found that when performing the heat treatmentduring a time period of 10 s to 2 hours at a pH below 5.4, curd isobtained, while at a pH exceeding 6.8, soluble whey protein results (seeFIG. 1). Thus, only in this rather narrow pH window will whey proteinsmicelles having a diameter of less than 1 μm be obtained. These micelleswill have an overall negative charge. The same micelle form can also beobtained symmetrically below the isoelectrical pH, i.e from 3.5 to 5.0,more preferably 3.8 to 4.5, resulting in micelles being positivelycharged (see FIG. 6).

Thus, according to an embodiment, in order to obtain positively chargedmicelles, micellisation of whey proteins may be done in a salt freesolution at a pH value adjusted between 3.8 and 4.5 depending on themineral content of the protein source.

Preferably, the micelles obtained will have an overall negative charge.Thus, in a preferred embodiment, the pH is adjusted to a range of from6.3 to 9.0, for a content in divalent cations comprised between 0.2% and2.5% in whey protein powder.

More specifically, to obtain negatively charged micelles, the pH isadjusted to a range of from 5.6 to 6.4, more preferably from 5.8 to 6.0for a low divalent cation content (e.g. less than 0.2% of the initialwhey protein powder). The pH may be increased up to 8.4 depending on themineral content of whey protein source (concentrate or isolate). Inparticular, the pH may be between 7.5 to 8.4, preferably 7.6 to 8.0 toobtain negatively charged micelles in the presence of large amounts offree minerals and the pH may be between 6.4 to 7.4, preferably 6.6 to7.2 to obtain negatively charged micelles in the presence of moderateamounts of free minerals. As a general rule, the higher the calciumand/or magnesium content of the initial whey protein powder, the higherthe pH of micellisation.

In order to standardize the conditions of formation of the whey proteinmicelles, it is most preferable to demineralise by any of the knowndemineralisation techniques (dialysis, ultrafiltration, reverse osmosis,ion exchange chromatography . . . ), any source of liquid native wheyproteins with a protein concentration ranging from that of sweet whey,microfiltration permeate of milk or acid whey (0.6% protein content) tothat of a concentrate at 30% protein content. The dialysis can be doneagainst water (distilled, deionised or soft), but as this will onlyallow removal of the ions weakly bound to the whey proteins, it is morepreferable to dialyse against an acid at pH below 4.0 (organic orinorganic) to better control the ionic composition of the whey proteins.By doing so, the pH of whey protein micelle formation will be below pH7.0, more preferably comprised between 5.8 to 6.6.

Prior to heating the whey protein aqueous solution, the pH is generallyadjusted by the addition of acid, which is preferably food grade, suchas e.g. hydrochloric acid, phosphoric acid, acetic acid, citric acid,gluconic acid or lactic acid. When the mineral content is high, the pHis generally adjusted by the addition of alkaline solution, which ispreferably food grade, such as sodium hydroxide, potassium hydroxide orammonium hydroxide.

Alternatively, if no pH adjustment step is desired, it is possible toadjust the ionic strength of the whey protein preparation while keepingthe pH constant. Then, ionic strength may be adjusted by organic orinorganic ions in such a way that allows micellisation at a constant pHvalue of 7. FIG. 4 represents an embodiment of the present invention,whereby micelles may be formed at a constant pH value of 7.0 while theionic strength is varied by the addition of 70-80 mM of arginine HCl.

A buffer may be further added to the aqueous solution of whey protein soas to avoid a substantial change of the pH value during heat treatmentof the whey protein. In principle, the buffer may be selected from anyfood-grade buffer system, i.e. acetic acid and its salts, such as e.g.sodium acetate or potassium acetate, phosphoric acid and salts thereof,e.g. NaH₂PO₄, Na₂HPO₄, KH₂PO₄, K₂HPO₄, or citric acid and salts thereofetc.

Adjusting the pH and/or the ionic strength of the aqueous solution,according to the present invention, results in a controlled processyielding micelles having a size between 100 nm-900 nm, preferablybetween 100-700 nm, most preferably between 200-400 nm. Preferably, theproportion of micelles with an average size comprised between 100-700 nmis greater than 80% when carrying out the process of the invention (seeFIG. 14).

In order to obtain regular shape micelles, it is also important,according to the invention, that the whey protein does not undergo anyhydrolysation step prior to micelle formation.

In a second step of the process of the present invention, the startingwhey protein aqueous solution is then subjected to the heat treatment.In this respect it has been found that for obtaining whey proteinmicelles, it is important to have the temperature in the range of fromabout 70 to below 95° C., preferably from 80 to about 90° C., morepreferably of from about 82 to about 89° C., even more preferably offrom about 84 to about 87° C., most preferred at about 85° C. It hasalso been found that, on an industrial scale, it is important that thetemperature be preferably less than 95° C., more preferably between 80°C. and 90° C., most preferably about 85° C.

Once the desired temperature has been reached, it is kept at thistemperature for a minimum of 10 seconds and a maximum of 2 hours.Preferably, the time period during which the aqueous whey proteinsolution is kept at the desired temperature ranges from 12 to 25minutes, more preferably from 12 to 20 minutes, or most preferably about15 minutes.

The heat treatment may also be achieved in a microwave oven or anysimilar equipment allowing heating by microwaves with a time/quantityratio of 10 s/10 mL for a 4 wt % protein solution heated in a 1500 Wapparatus up to boiling temperature (98° C. at an altitude of 833 m). Acontinuous process may also be used by addition of 8 or more magnetronsaround a glass tube potentially prolonged by a holding tube to increasethe time of incubation.

As shown in FIG. 2, turbidity measurements are an indication of micelleformation. According to the present invention, the turbidity measured byabsorbance at 500 nm is at least 3 absorbance units for 1% proteinsolution but can reach 16 absorbance units when the yield ofmicellisation is above 80% (see FIG. 2).

To further illustrate the effect of micelle formation from aphysicochemical point of view, a 1 wt % dispersion of Bipro® has beenheated for 15 minutes at 85° C. at pH 6.0 and 6.8 in MilliQ water. Thehydrodynamic diameter of the aggregates obtained after heat treatmentwas measured by dynamic light scattering. The apparent molecular weightof the aggregates was determined by static light scattering using theso-called Debye plot. The surface hydrophobicity was probed using thehydrophobic ANS probe and the free accessible thiol groups by the DTNBmethod using cystein as the standard amino acid. Finally, the morphologyof the aggregates was studied by negative staining TEM. The results arepresented in table 1.

TABLE 1 Physicochemical properties of soluble whey protein aggregatesobtained by heat treatment (85° C., 15 min) of a 1 wt % proteindispersion in presence or absence of NaCl. protein accessible surface SHgroups hydrodynamic molecular ζ- hydrophobicity (nmol diameter weightM_(w) potential (μg · mmol⁻¹ SH · mg⁻¹ pH (nm) (×10⁶ g · mol⁻¹)morphology (mV) ANS) prot.) 6.0 120.3 ± 9.1 27.02 ± 8.09 Spherical −31.8± 0.8 105.4 3.5 ± 0.4 micelles 6.8  56.2 ± 4.6 0.64 ± 0.01 linear −27.9± 1.2 200.8 6.8 ± 0.5 aggregates

From table 1, it is clear that the whey protein micelles that wereformed at pH 6.0 allow protein to decrease its specific ANS surfacehydrophobicity by a factor of 2 compared to non-micellised whey proteinheated in the same condition, but at pH 6.8. The micelle formation canbe also seen on the very high molecular weight of 27×10⁶ g.mol⁻¹compared to 0.64×10⁶ g.mol⁻¹ for non-micellised protein, indicating avery condensed state of the matter within the micelle (low amount ofwater). Interestingly enough, the ζ-potential of the micelles is evenmore negative than the non-micellised proteins even if the latter havebeen formed at a more basic pH than the micelles. This is the result ofa more hydrophilic surface of the micelles being exposed to the solvent.Finally, one should note that the thiol reactivity of the micelles ismuch lower than that of the non-micellised protein because of thedifferent pH of heat treatment.

It has been found that the conversion yield of native whey protein tomicelles decreases when the initial protein concentration is increasedbefore pH adjustment and heat treatment. For example, when starting witha whey protein isolate Prolacta 90 (lot 673 from Lactalis), the yield offormation of whey protein micelles drops from 85% (when starting with 4%proteins) to 50% (when starting with 12% of proteins). In order tomaximize the formation of whey protein micelles (>85% of the initialprotein content), it is better to start with an aqueous whey proteinsolution having a protein concentration below 12%, preferably below 4%.Depending on the intended final applications, the protein concentrationis adjusted before heat treatment to manage the optimal whey proteinmicelles yield.

The whey proteins micelles obtained according to the present methodshall have an average size of less than 1 μm, preferably of from 100 to900 nm, more preferably from 100 to 700 nm, most preferably from 200-400nm.

Depending on the desired application, the yield of micelles beforeconcentration is at least 35%, preferably at least 50%, more preferablyat least 80% and the residual soluble aggregates or soluble proteincontent is preferably below 20%. The average micelle size ischaracterised by a polydispersity index below 0.200. It has beenobserved that whey protein micelles could form aggregates around pH 4.5,with however no sign of macroscopic phase separation after at least 12hours at 4° C.

The purity of whey protein micelles produced according to the method ofthe present invention can be obtained by determining the amount ofresidual soluble proteins. Micelles are eliminated by centrifugation at20° C. and 26900 g for 15 min. The supernatant is used to determine theprotein amount in quartz cuvettes at 280 nm (1 cm light pathlength).Values are expressed as a percentage of the initial value before heattreatment.Proportion of micelles=(Amount of initial proteins−amount of solubleproteins)/Amount of initial proteins

An advantage of the method of the present invention is that the wheyprotein micelles prepared accordingly have not been submitted to anymechanical stress leading to reduction of the particle size duringformation, contrary to conventional processes. This method inducesspontaneous micellisation of whey proteins during heat treatment in theabsence of shearing.

The whey protein micelles may be used as such in any composition, suchas nutritional compositions, cosmetic compositions, pharmaceuticalcompositions etc. Furthermore, the whey protein micelles may be filledwith an active component. Said component may be selected from coffee,caffeine, green tea extracts, plant extracts, vitamins, minerals,bioactive agents, salt, sugar, sweeteners, aroma, fatty acids, oils,protein hydrolysates, peptides etc. and mixtures thereof.

Furthermore, the whey protein micelles (pure or filled with activecomponents) of the present invention may be coated with an emulsifiersuch as phospholipids, for example, or other coating agents such as aprotein, a peptide, a protein hydrolysate or a gum such as acacia gum inorder to modulate the functionality and the taste of the whey proteinmicelles. When a protein is used as a coating agent, it may be selectedfrom any proteins having an isoelectric point significantly higher orlower than whey protein. These are, for example, protamine, lactoferrinand some rice proteins. When a protein hydrolysate is used as coatingagent, it is preferably a hydrolysate from proteins such as protamine,lactoferrin, rice, casein, whey, wheat, soy protein or mixtures thereof.Preferably, the coating is an emulsifier selected from sulphated butyloleate, diacetyltartaric acid esters of mono- and diglycerides, citricacid esters of monoglycerides, stearoyl lactylates and mixtures thereof.FIG. 17 is a schematic representation of such coating with sulphatedbutyl oleate. Coating may be carried out by any methods known in theart. Furthermore, co-spraydrying, as described further herein, may alsoresult in a coating of the whey protein micelles.

The whey protein micelles have shown to be ideally suited for use as anemulsifier, fat substitute, substitute for micellar casein or foamingagent, since they are able to stabilize fat and/or air in an aqueoussystem for prolonged period.

The foam stability is shown in FIG. 5 which compares the use ofnon-micellised whey protein versus the micellised whey protein of thepresent invention.

Thus, whey protein micelles may be used as an emulsifying agent, forwhich the material is ideally suited, since it has a neutral taste andno off-flavour is created by the use of such material. They may also beused as micellar casein substitute.

In addition, the present whey protein micelles are still in a conditionto serve as whitening agent, so that with one compound several tasks maybe fulfilled. Since whey is a material abundantly available, the usethereof reduces the cost of a product requiring an emulsifying, filling,whitening or foaming agent, while at the same time adding to itsnutritional value.

Accordingly, the whey protein micelles obtained according to the methodof the present invention can be used for the preparation of any kind ofconsumable product requiring stabilisation of an emulsion or a foam,such as e.g. present in mousse or ice cream, in coffee creamers, or alsoin low fat or essentially fat free dairy products, or also where itfinds application as a micellar casein substitute. By “consumable” ismeant any food product in any form, including beverages, soups,semi-solid foods etc. which can be consumed by a human or an animal.Examples of products, where the present whey protein micelles may findapplication are for example, dairy products, mayonnaise, salad dressing,pasteurized UHT milk, sweet condensed milk, yoghurt, fermented milks,sauces, reduced fat sauces such as béchamel sauce for instance,milk-based fermented products, milk chocolate, white chocolate, darkchocolate, mousses, foams, emulsions, ice creams, fermented cereal basedproducts, milk based powders, infant formula, diet fortifications, petfood, tablets, liquid bacterial suspensions, dried oral supplement, wetoral supplement, performance nutrition bars, spreads, fruit drinks,coffee mixes.

Furthermore, the present whey protein micelles may be used either aloneor together with other active materials, such as polysaccharides (e.g.acacia gum or carrageenans) to stabilise matrices and for example milkyfoam matrices. Due to their neutral taste, their whitening power andtheir stability after heat treatment, the present whey proteins micellesmay be used to increase skimmed milk whiteness and mouth feel.

As well as increasing the whitening power of dairy systems for the sametotal protein content, the fat content in a food matrix may be reduced.This feature represents a particular advantage of the present wheyprotein micelles, since it allows producing low-fat products, forexample adding a milk creamer without adding additional fat derived fromthe milk as such.

In the method of the present invention, the whey protein micelledispersion obtained after heat treatment is concentrated to yield a wheyprotein micelle concentrate.

Accordingly the concentration step may be carried out by evaporation,centrifugation, sedimentation, ultrafiltration and/or bymicrofiltration.

Evaporation may be carried out on the micelles dispersion by feeding itto an evaporator under vacuum, having a temperature between 50° C. and85° C.

Centrifugation may be carried out with high acceleration rate (more than2000 g) or low acceleration rate (less than 500 g) after acidificationof the whey protein micelle dispersion at a pH lower than 5, preferably4.5.

Spontaneous sedimentation may also be carried out on the whey proteinmicelle dispersion by acidification. Preferably, the pH will be 4.5 andthe sedimentation time is more than 12 hours.

Preferably, concentration of the whey protein micelles according to thepresent invention may be achieved by microfiltration of the micellesdispersion. This enriching technique not only enables to concentratewhey protein micelles by removing the solvent but also enables theremoval of non-micellised protein (such as native proteins or solubleaggregates). Thus, the final product only consists of micelles (aschecked by Transmission Electron Microscopy—cf. FIGS. 9 and 10). In thiscase, the concentration factor that is possible to achieve is obtainedafter the initial flow rate of permeate through the membrane has droppedto 20% of its initial value.

The whey protein concentrate obtained by the method of the presentinvention will have a protein concentration of at least 12%.Furthermore, the concentrate will contain at least 50% of the protein inthe form of micelles.

It is interesting to note that the concentrate, if adjusted to a proteincontent of 10% has the ability to withstand a subsequent heat treatmentat 85° C. for 15 min at pH 7.0 in presence for example of up to 0.15 Mof sodium chloride, as shown in FIG. 11. As a matter of comparison, anative whey protein dispersion (Prolacta90, lot 500658 from Lactalis)forms a gel in the presence of 0.1 M of sodium chloride at a proteinconcentration of only 4% (cf. FIG. 12).

The present invention also presents the benefit that the high stabilityof the micelle structure is preserved during the concentration step.Furthermore, the micelles according to the present invention have aProtein Efficiency Ratio equivalent to the starting whey protein of atleast 100, preferably at least 110, which makes them importantnutritional ingredients.

The enrichment of the whey protein micelles offers the exceptionaladvantages that protein-enriched products may be obtained atconcentration previously not attainable. Furthermore, since theconcentrate may act as a fat substitute while maintaining desirablestructural, textural and organoleptic properties, a wider variety oflow-fat product may be obtained.

Additionally, it presents the cost advantage that a smaller amount ofconcentrate is needed to obtain the desired effects.

The whey protein micelle concentrate (from evaporation ormicrofiltration) can be used in liquid form as a dispersion or insemi-solid form, or in a dried form. It may be used in a great varietyof applications such as those described above with respect to the wheyprotein micelles applications.

For instance, the 20% protein concentrate obtained by evaporation has acreamy, semi-solid texture (see FIG. 18) and can be texturised in aspreadable texture by acidification using lactic acid. This liquid,creamy, pasty texture can be used to prepare acid, sweet, salty,aromatic, protein-rich consumables.

The whey protein micelles concentrate in any form may be mixed with 5%of an acidic fruit base and 5% of sucrose in order to obtain a stablewhey protein enriched acidic fruit drink. It may also be used in themanufacture of milk products, ice cream, or used as coffee whiteneramongst others.

Further applications include skin care and mouth care, such astoothpaste, chewing gum, or gum-cleaning agent for instance.

The whitening power of the concentrate in any form is tremendouslyincreased in comparison to the non-concentrated micelles or to thenative protein powders. For example, the whitening power of 4 mL of a15% whey protein micelle concentrate is equivalent to 0.3% of titaniumoxide in 100 mL of a 2% soluble coffee cup. Interestingly, it ispossible to disperse soluble coffee and sucrose into a whey proteinmicelle concentrate so that a 3-in-1 concentrate having a total solidsconcentration of 60% without fat is obtained.

The concentrate may be used as such or diluted depending on theapplication.

For instance, the whey protein micelle concentrate in liquid or driedform may be diluted to a protein content of 9% like in sweet andcondensed milk. The milk minerals, lactose and sucrose can be added sothat the final product will have similar nutritional profile compared tomilk, but only whey protein as the protein source. This whey proteinbased blend is more stable than sweet condensed milk against Maillardreaction (based on the speed of development of a brown colour) whenincubated 2 hours at 98° C. (temperature of boiling water at an altitudeof 833 m).

The dried form of the whey protein concentrate obtained by the method ofthe present invention may be obtained by any known techniques, such asspray-drying, freeze-drying, roller drying etc. Thus, the whey proteinconcentrate of the present invention may be spray-dried with or withoutaddition of further ingredients and may be used as a delivery system ora building block to be used in a wide range of processes, e.g.consumables production, cosmetic applications etc.

FIG. 8 shows a powder obtained by spray-drying without addition of anyfurther ingredients, having an average particle diameter size greaterthan 1 micron due to the micelle aggregation occurring duringspray-drying. A typical average volume median diameter (D₄₃) of thepowders of the invention is between 45 and 55 microns, preferably 51microns. The surface median diameter (D₃₂) of the powders of the presentinvention is preferably between 3 and 4 microns, more preferably it is3.8 microns.

The moisture content of the powders obtained after spray-drying ispreferably less than 10%, more preferably less than 4%.

Such a whey protein micelle powder may comprise at least 85% wheyprotein, from which at least 20%, preferably more than 50%, mostpreferably more than 80% are in the micellar form.

Furthermore, the whey protein micelles powder of the present inventionhave a high binding capacity for solvents such as water, glycerol,ethanol, oil, organic solvents etc. The binding capacity of the powdersto water is at least 50%, preferably at least 90%, most preferably atleast 100%. For solvents such as glycerol and ethanol, the bindingcapacity is of at least 50%. For oil, the binding capacity is at least30%. This property of the whey protein micelle powders of the presentinvention allows these to be sprayed or filled with further functionalingredients such as coffee, caffeine, green tea extracts, plantextracts, vitamins, minerals, bioactive agents, salt, sugar, sweeteners,aroma, fatty acids, oils, protein hydrolysates, peptides etc. andmixtures thereof.

The functional ingredients may be included in the powder in an amount of0.1-50%. Thus, the powder may act as a carrier for those functionalingredients. This presents the advantage that, for instance, caffeinebitterness perception is reduced when filled into the powders of thepresent invention and used in caffeinated nutrition bars for instance.

Additional ingredients may be mixed to the whey protein micelleconcentrate prior to spray-drying. These comprise soluble or non-solublesalts, peptides, protein hydrolysates e.g. cultured wheat glutenhydrolysate for example, probiotic bacteria, stains, sugars,maltodextrins, fats, emulsifiers, sweeteners, aroma, plant extracts,ligands, bioactive agents, caffeine, vitamins, minerals, drugs, milk,milk proteins, skimmed milk powder, micellar casein, caseinate, vegetalprotein, amino acids, polyphenols, pigment etc. and any possiblemixtures thereof. The resulting mixed whey protein micelle powderscomprise whey protein micelles and at least one additional ingredient ina weight ratio ranging from 30:1 to 1:1000.

This co-spraydrying results in powders consisting of whey proteinmicelles agglomerated or coated with an additional ingredient.Preferably, the weight ratio of whey protein micelles to additionalingredient is 1:1. This may further facilitate solubilisation of thesepowders and may be of particular interest in the manufacture ofdehydrated food products such as soups, sauces etc. comprising wheyprotein micelles.

The whey protein micelle powders obtained by the present invention arecharacterised by an internal structure composed mainly of hollow spheresbut also of collapsed spheres (cf. FIG. 19). The hollow spheresstructure can be easily explained by the formation of the vapour dropletwithin the WPM concentrate droplet during the spray drying. As thevapour droplet left the WPM droplet due to a temperature above 100° C.,a hollow sphere remained. The “bone-shape” is due to a combination ofthe water evaporation from droplet and the external pressure within thedroplet.

The internal structure of the spherical hollow spheres was investigatedby SEM after sectioning the particle close to its diameter (FIG. 20,left). The wall thickness of the particle was around 5 μm and seemedvery smooth, whereas the inner structure had a more grainy appearance.Increased magnification showed that this graininess was in fact due tothe presence of the initial WPM that were fused to form the inner matrixof the powder particle. Interestingly, the spherical shape of themicelles was kept during spray drying as well the homogeneous particlesize distribution (FIG. 20, right).

Thus, on a microscopic basis, whey protein micelle powders arecharacterised by a unique granule morphology of hollow or collapsedspheres containing intact and individualised whey protein micelles.

Whey protein micelle powders are characterised by a very highflowability, which offers advantages not previously obtainable. Forinstance, these powders behave almost as liquids and present theadvantages of easy usability and transferability. The angle of repose ofthese powders is preferably below 35°, more preferably below 30°. Such alow angle of repose allows the powders of the present invention to beused as flowing agents in food applications, for instance.

A very important feature of these powders, mixed or “pure” is that thebasic micelle structure of the whey proteins is conserved. FIG. 15 showsa whey protein powder grain which has been sectioned, and whereby theindividual whey protein micelles are observable. Furthermore, themicelle structure can be easily reconstituted in solvents. It has beenshown that the powders obtained from whey protein micelle concentratecan be easily redispersed in water at room temperature or at 50° C. Thesize and structure of the whey protein micelles are fully conservedcompared to the initial concentrate. For example, in FIG. 13, the wheyprotein concentrate that was spray-dried at 20% protein concentrationhas been redispersed in deionised water at 50° C. at a proteinconcentration of 4%. The structure of the micelles has been probed byTEM and can be compared to FIG. 10. A similar shape of micelles wasobtained. The diameter of the micelles was found to be 315 nm by dynamiclight scattering with a polydispersity index of 0.2. FIG. 16 also showsdispersion of a freeze-dried whey protein micelle powder, wherein themicelles are reconstituted.

The fact that the whey protein micelles and only a minor aggregatedfraction were observed in solution after reconstitution of thespray-dried or freeze-dried powder confirms that whey protein micellesare physically stable regarding spray-drying and freeze-drying.

The powders of the present invention may be used in a wide range ofapplications, such as all those described above in relation to wheyprotein micelles and the concentrates thereof. For instance,protein-enriched consumables, such as chocolate, performance nutritionbars, dehydrated culinary products, chewing-gum etc. can be easilyproduced by using the micelle concentrate powders.

Due to their high stability to processing, the powders of the presentinvention may also be further coated by emulsifiers or gums, forinstance. This may be advantageous to modulate the functionality and thetaste of these powders.

The following examples illustrate the present invention without limitingit thereto.

EXAMPLES

The invention is further defined by reference to the following examplesdescribing in detail the preparation of the micelles of the presentinvention. The invention described and claimed herein is not to belimited in scope by the specific embodiments herein disclosed, sincethese embodiments are intended as illustrations of several aspects ofthe invention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

Example 1 Micellisation of β-Lactoglobulin by pH Adjustment

β-Lactoglobulin (lot JE002-8-922, 13-12-2000) was obtained from Davisco(Le Sueur, Minn., USA). The protein was purified from sweet whey byultra-filtration and ion exchange chromatography. The composition of thepowder is 89.7% protein, 8.85% moisture, 1.36% ash (0.079% Ca²⁺, 0.013%Mg²⁺, 0.097% K⁺, 0.576% Na⁺, 0.050% Cl⁻. All other reagents used were ofanalytical grade (Merck Darmstadt, Germany).

The protein solution was prepared at 0.2% concentration by solvation ofβ-lactoglobulin in MilliQ® water (Millipore), and stirring at 20° C. for2 h. Then pH of aliquots was adjusted to 5.0, 5.2, 5.4, 5.6, 5.8, 6.0,6.2, 6.4, 6.6, 6.8, 7.0 by HCl addition. The solutions were filled in 20ml glass vials (Agilent Technologies) and sealed with aluminum capsulescontaining a silicon/PTFE sealing. The solutions were heated at 85° C.for 15 min (time to reach the temperature 2.30-3.00 min). After the heattreatment, the samples were cooled in ice water to 20° C.

The visual aspect of products (FIG. 1) indicates that the optimal pH ofmicellisation is 5.8.

Example 2 Micellisation of Whey Protein Isolate

Whey protein isolate (WPI) (Bipro®, Batch JE032-1-420) was obtained fromDavisco (Le Sueur, Minn., USA). The composition of the powder isreported in table 2.

The protein solution was prepared at 3.4% protein by solvation of wheyprotein powder in MilliQ® water (Millipore), and stirring at 20° C. for2 h. The initial pH was 7.2. Then pH of aliquots was adjusted at 5.6,5.8, 6.0, 6.2, 6.4 and 6.6 by HCl 0.1N addition.

The solutions were filled in 20 ml glass vials (Agilent Technologies)and sealed with aluminum capsules containing a silicon/PTFE sealing. Thesolutions were heated at 85° C. for 15 min (time to reach thetemperature 2.30-2.50 min). After the heat treatment, samples werecooled in ice water to 20° C.

The turbidity of heated whey proteins has been determined at 500 nm and25° C., samples were diluted to allow the measurement in the range of0.1-3 Abs unit (Spectrophotometer Uvikon 810, Kontron Instrument).Values were calculated for the initial protein concentration 3.4%.

The pH of micellisation was considered to be reached upon stability(less than 5% variation of the initial value) of the absorbance measuredat 500 nm within an interval of 10 minutes for the same sample asillustrated by the FIG. 2. For this product the optimal pH formicellisation was 6.0 to 6.2. For this pH adjusted before heat treatmentstable turbidity was 21 and residual soluble protein evaluated byabsorbance at 280 nm after centrifugation was 1.9%. We can conclude that45% of initial proteins were transformed in micelles at pH 6.0.

TABLE 2 Composition of WPI and sample characteristics aftermicellisation Supplier Davisco Product name Bipro Batch number JE032-1-420 Composition (mg/100 g) Sodium 650 Potassium 44 Chloride*10 if≦40 10 Calcium 82 Phosphorus 49 Magnesium 6 Initial pH 7.2 pHmicellisation 6.0 Turbidity (500 nm) for 3.4% protein in 21 solutionResidual Soluble protein (%) by 1.9 absorbance at 280 nm

Example 3 Microscopic Observation of Micelles

Production of Micelles:

Protein solution was prepared at 2% protein by salvation of whey proteinpowder (WPI 90 batch 989/2, Lactalis, Retier, France) in MilliQ® water(Millipore), and stirred at 20° C. for 2 h. Then pHs of aliquots wereadjusted using HCl 0.1N or NaOH 0.1N.

The solutions were filled in 20 ml glass vials (Agilent Technologies)and sealed with aluminum capsules containing a silicon/PTFE sealing. Thesolutions were heated at 85° C. for 15 min (time to reach thetemperature 2.30-2.50 min). After the heat treatment, the samples werecooled in ice water to 20° C. For this product the optimal pH formicellisation was 7.4.

Microscopic Observations:

Liquid micelle samples were encapsulated in agar gel tubes. Fixation wasachieved by immersion in a solution of 2.5% glutaraldehyde in 0.1M, pH7.4 cacodylate buffer and post-fixation with 2% Osmium tetroxide in thesame buffer, both solutions containing 0.04% Ruthenium red. Afterdehydration in a graded ethanol series (70, 80, 90, 96, 100% ethanol),the samples were embedded in Spurr resin (Spurr/ethanol 1:1, 2:1, 100%).After polymerization of the resin (70° C., 48 hours), semi-thin andultra-thin sections were cut with a Leica ultracut UCT ultra-microtome.Ultra-thin sections, stained with aqueous uranyl-acetate and leadcitrate, were examined in transmission electron microscopy (PhilipsCM12, 80 kV).

TEM micrograph is presented in FIG. 3. Obtained micelles are presentinga spherical shape with a diameter of 200 nm.

Particle Size Distribution

The intensity-based size distributions of micelles were measured forthose micelles obtained by heat-treatment of a 1 wt % β-lactoglobulindispersion for 15 min at 85° C. at pH 4.25 (positively charged with azeta potential around +25 mV) and at pH 6.0 (negatively charged with azeta potential around −30 mV). Z-averaged hydrodynamic diameter of themicelles was 229.3 mm at pH 4.25 an 227.2 at pH 6.0. β-LG and wheyprotein aggregations were followed using dynamic light scattering. ANanosizer ZS apparatus (Malvern Instruments, UK) equipped with a laseremitting at 633 nm and with 4.0 mW power was used. The instrument wasused in the backscattering configuration, where detection is done at ascattering angle of 173°. This allows considerable reduction of themultiple scattering signals found in turbid samples. Samples were placedin a squared quartz cell (Hellma, pathlength 1 cm). The path length ofthe light beam was automatically set by the apparatus, depending on thesample turbidity (attenuation). The autocorrelation function wascalculated from the fluctuation of the scattered intensity). The resultsare presented in FIG. 6. It shows that the average particle ischaracterized by a very narrow polydispersity index (<0.200).

Example 4 Micellisation of a β-Lactoglobulin at a Constant pH

The method described in example 1 was repeated using an aqueous solutionof 2% β-lactoglobulin. The pH of this solution has been adjusted to 7.0after adding Arginine HCl solutions to obtain a final salt concentrationranging from 5 to 200 mM and a final β-lactoglobulin concentration of1%. Subsequent heat treatment (80° C., 10 min, about 2 min heating up)was carried out to produce micelles.

The results are shown in FIG. 4 and clearly indicate that only in theionic strength range of from about 50 to 70 mM, a substantial turbiditycan be observed, indicating the presence of whey protein micelles.

Example 5 Preparing a Whitening Agent

Native whey proteins (WPI 95 batch 848, Lactalis; 8 wt-% aqueoussolution) were treated according to example 2. The resulting productlightness (L) was measured in trans-reflectance mode using a MacBethCE-XTH D65 10° SCE apparatus equipped with a 2 mm measuring cell. Theresulting lightness was L=74.8, that could be compared to the value ofL=74.5 for full-fat milk.

Example 6 Preparing a Coffee Creamer

Native whey proteins (Bipro®, lot JE 032-1-420, 0.5 wt-% aqueoussolution) were mixed at 50° C. with 10 wt.-% partially hydrogenated palmoil, 14 wt. % maltodextrin (DE 21) and in presence of 50 mMphosphate-citrate buffer adjusted to the micellisation pH of 6.0 forthis Bipro®. The mixture was homogenized under 400/50 bars using aRannie homogeniser and subsequently heat-treated for 15 minutes at 85°C.

The emulsion obtained showed a high stability over a time period of atleast one month at the conditions of storage at 4° C. and gave awhiteness of L=78 compared to a reference liquid creamer (Crème à Café,Emmi, Switzerland) having a fat content of 15% and a lightness ofL=75.9.

Example 7 Preparing an Aqueous Foam

Native β-lactoglobulin (Biopure, Davisco, lot JE 002-8-922, 2 wt-%aqueous solution) was mixed with 120 mM Arginine HCl solution so thatthe final β-lactoglobulin concentration was 1 wt. % and Arginine HCl 60mM. The pH was then adjusted to 7.0 by addition of 1N HCl. The mixturewas then heat treated at 80° C. for 10 minutes so that 90% of initialβ-lactoglobulin was converted into micelles having a z-averaged diameterof 130 nm. In this case, the diameter of the micelles was determinedusing a Nanosizer ZS apparatus (Malvern Instruments, UK). The sample waspoured in a quartz cuvette and variations of the scattered light wererecorded automatically. The obtained autocorrelation function was fittedusing the cumulants method so that the diffusion coefficient of theparticles could be calculated and thereafter the z-averaged hydrodynamicdiameter using the Stokes-Einstein law. For this measurement, therefractive index of the solvent was taken as 1.33 and that of themicelles 1.45. A volume of 50 mL of the resulting dispersion ofβ-lactoglobulin micelles is then foamed by nitrogen sparging through aglass frit generating bubbles of 12-16 μm to produce a foam volume of180 cm³ using the standardised Foamscan™ (ITConcept) apparatus. Thevolume stability of the foam was then followed with time at 26° C. usingimage analysis and compared to the stability of the foam obtained withβ-lactoglobulin treated in the same conditions, but without ArginineHCl, where no micelles were formed. FIG. 5 shows that the foam volumestability is greatly improved by the presence of β-lactoglobulinmicelles.

Example 8 Whey Based Fermented Dairy Product Fermentation Trials

Material

Whey protein isolate (WPI) (Bipro®) was obtained from Davisco (Le Sueur,Minn., USA) (protein concentration 92.7%). Spray dried whey permeate(Variolac 836): Lactose concentration: 83%-Minerals: 8%

Lactic Acid 50%

Edible Lactose (Lactalis)

De-ionized water

Method

The Bipro® powder was dissolved in de-ionized water in order to have aprotein concentration of 4.6%, i.e. for 3 liters of solution 154.5 g ofWPI powder and 2845.5 g of water. The hydration time was 3 hours. Afterhydration, this solution has been divided in samples of 200 ml toprepare the different trials:

TABLE 3 Whey Heating permeate pH 85° C./15 Trial (%) Lactose (%)adjustment min 1 2.9 2.5 6.5 + 2 0 5 6 + 3 0 5 6.7 − 4 0 5 6.7 + 5 0 56.1 + 6 0 0 6 + 7 0 5 (added after pH 6 − adjustment) 8 0 5 (added afterpH 6 + adjustment)

For each solution, lactic acid at 50% has been added to adjust the pHbefore heating.

Samples were heated with the double boiler up to 85° C. and maintain atthis temperature during 15 minutes. After heating, solutions were cooledat 40° C. and inoculated with Lactobacillus bulgaricus and Streptococcusthermophilus. Samples were incubated 5 h30 in a steam room at 41° C.before to be placed in a cold room at 6° C.

The results are presented in Table 4.

TABLE 4 pH Whey after Trial permeate Lactose pH Heating 5 h 30 Aspect1 + + 6.5 + 4.68 Very firm 2 − + 6 + 4.7 Firm 3 − + 6.7 − 5.78 Liquid 4− + 6.7 + 4.81 Very firm 5 − + 6.1 + 4.59 Very firm 6 − − 6 + 4.99 Veryfirm 7 − − added 6 − 4.87 Liquid after pH with adjustment white speckles8 − − added 6 + 4.77 Firm after pH adjustment

Example 9 Whey Protein Boosted Ice Cream with Reduced Fat Content

Material

Whey protein isolate (WPI, Prolacta90® from Lactalis, Rëtiers, France)with a protein content of 90% Skim milk powder with 35% protein content

Sucrose

Maltodextrins DE39

Anhydrous milk fat

Emulsifier

De-ionised water

Edible hydrochloric acid 1M

Method

Using a double-jacketed 80 L tank, the Prolacta90® powder was dispersedat 50° C. in de-ionized water at a protein concentration of 9.67 wt %under gentle stirring in order to avoid foam formation, i.e. 3.3 kg ofProlacta90® were dispersed in 31.05 kg of de-ionised water. After 1 hourof dispersion, the pH of the dispersion was adjusted to themicellisation pH by addition of HCl. The temperature of the dispersionwas raised to 85° C. and maintained for 15 minutes in order to generatethe whey protein micelles. After 15 minutes, the temperature wasdecreased to 50° C. and the additional ingredients were sequentiallyadded to the micelles dispersion (i.e. skim milk powder, maltodextrinsDE39, sucrose, emulsifier and anhydrous milk fat). The final amount ofmix was 50 kg with total solids content of 39.5% and a fat content of 5wt %. After 30 minutes of hydration, the mix was two-step homogenised(80/20 bars) and pasteurised (86° C./30 s) before ageing duringovernight. The day after, the ice-cream mix was frozen at an overrun of100% using a Hoyer MF50 apparatus and hardened at −40° C. before storageat −20° C. The final ice cream contained 8 wt % proteins (20% caseins,80% whey proteins) and 5 wt % fat on the ice cream mix basis.

Example 10 Powdered Whey Protein Micelles Obtained by Spray-Drying

Material

Whey protein isolate (WPI, Prolacta90® from Lactalis, Rétiers, France)with a protein content of 90%

Edible lactose

Maltodextrins DE39

De-ionised water

Edible hydrochloric acid 1M

Method

Using a double-jacketed 100 L tank, the Prolacta90® powder was dispersedat 50° C. in de-ionized water at a protein concentration of 10 wt %under gentle stirring in order to avoid foam formation, i.e. 11 kg ofProlacta90® were dispersed in 89 kg of de-ionised water. After 1 hour ofdispersion, the pH of the dispersion was adjusted to the micellisationpH (around 6.3 in that case) by addition of HCl. The temperature of thedispersion was raised to 85° C. and maintained for 15 minutes in orderto generate the whey protein micelles. After 15 minutes, the temperaturewas decreased to 50° C. and the 10 wt % whey protein micelles dispersionwas split in two batches of 50 kg. In a first trial, 20 kg of lactosewere dispersed in 50 kg of micelles dispersion at 50° C. and stirred for30 min. Similarly, 20 kg of maltodextrins DE39 were added to theremaining 50 kg of whey protein micelles dispersion.

The two mixtures were then spray dried into a NIRO SD6.3N tower at aflow rate of 15 L/h. The air input temperature was 140° C. and the airoutput temperature was 80° C. The water content of the obtained powderswas lower than 5%.

The size of the whey protein micelles was determined in presence oflactose and maltodextrin (DE39) in water using dynamic light scatteringbefore and after spray drying. The total protein concentration was setto 0.4 wt % by dilution of the dispersion before spray drying orreconstitution of the powder in order to be in the dilute regime ofviscosity for whey protein micelles. A Nanosizer ZS apparatus (MalvernInstruments) was used and micelle diameter was averaged from 20measurements.

The particle diameter determined for whey protein micelles in presenceof lactose and maltodextrins (DE39) was 310.4 nm and 306.6,respectively. After reconstitution of the powders, the respectivediameters were found to be 265.3 nm and 268.5, respectively. Thesemeasurements confirm than whey protein micelles were physically stableregarding spray drying. The results were corroborated by TEM microscopyobservations of 0.1 wt % whey protein micelles dispersions in waterusing negative staining in presence of 1% phosphotungstic acid at pH 7.A Philips CM12 transmission electron microscope operating at 80 kV wasused. Whey protein micelles were observed in solution before spraydrying and after reconstitution of the spray-dried powder. No differenceof morphology and structure could be detected.

Example 11 Concentration by Evaporation

A whey protein isolate Prolacta 90 from Lactalis (lot 500648) has beenreconstituted at 15° C. in soft water at a protein concentration of 4%to reach a final batch size of 2500 kg. The pH was adjusted by additionof 1M hydrochloric acid so that the final pH value was 5.90. The wheyprotein dispersion was pumped through plate-plate APV-mix heat exchangerat a flow rate of 500 l/h. Pre-heating at 60° C. was followed by heattreatment of 85° C. for 15 minutes. Formation of whey protein micelleswas checked by measurement of particle size using dynamic lightscattering as well a turbidity measurement at 500 nm. The obtained 4%whey protein micelles dispersion was characterised by a hydrodynamicradius of particles of 250 nm, a polydispersity index of 0.13 and aturbidity of 80. The whey protein micelle dispersion was then used tofeed a Scheffers evaporator at a flow rate of 500 l/h. The temperatureand vacuum in the evaporator were adapted so that around 500 kg wheyprotein micelles concentrate having a protein concentration 20% wereproduced and cooled down to 4° C.

Example 12 Enrichment by Microfiltration

A whey protein isolate Prolacta 90 from Lactalis (lo 500648) has beenreconstituted at 15° C. in soft water at a protein concentration of 4%to reach a final batch size of 2500 kg. The pH was adjusted by additionof 1M hydrochloric acid so that the final pH value was 5.90. The wheyprotein dispersion was pumped through plate-plate APV-mix heat exchangerat a flow rate of 500 L/h. A pre-heating at 60° C. was followed by heattreatment of 85° C. for 15 minutes. Formation of whey protein micelleswas checked by measurement of particle size using dynamic lightscattering as well a turbidity measurement at 500 nm. The obtained 4%whey protein micelles dispersion was characterised by a hydrodynamicradius of particles of 260 nm, a polydispersity index of 0.07 and aturbidity of 80. The micelle form of the protein was also checked byTEM, and micelle structures with an average diameter of 150-200 nm wereclearly visible (FIG. 9). The whey protein micelle dispersion could becooled at 4° C. for storage or directly used to feed a filtration unitequipped with a 6.8 m² Carbosep M14 membrane at a flow rate of 180 L/h.In that case, the concentration of the whey protein micelles wasperformed at 10 to 70° C. until the permeate flow rate reached 70 L/h.In that case, the final whey protein concentrate contained 20% ofproteins. The structure of the micelles in the concentrate was checkedby TEM, and clearly no significant change was visible compared to the 4%whey protein dispersion before microfiltration (FIG. 10).

Example 13 Whey Protein Micelles Powder Comprising at Least 90% WheyProtein

200 kg of a whey protein micelle concentrate obtained by microfiltrationat 20% protein (see example above) were injected in a Niro SD6.3N towerusing an atomisation nozzle (Ø=0.5 mm, spraying angle=65°, pressure=40bars) at a product flow rate of 25 kg/h. The inlet temperature ofproduct was 150° C. and the outlet temperature was 75° C. The airflow inthe tower was 150 m³/h. The moisture content in the powder was less than4% and the powder was characterized by a very high flowability. Scanningelectron microscopy of the powder exhibited very spherical particleshaving an apparent diameter ranging from 10 to 100 μm (FIG. 8).

Example 14 Mixed Whey Protein Micelle Powder

20 kg of a whey protein micelle concentrate were mixed with 1.7 kg ofmaltodextrins with a DE of 39 so that the final whey protein micelle tomaltodextrin ratio in powder is 70/30. This mixture was injected in aNiro SD6.3N tower using an atomisation nozzle (Ø=0.5 mm, sprayingangle=65°, pressure=40 bars) at a product flow rate of 25 kg/h. Theinlet temperature of product was 150° C. and the outlet temperature was75° C. The airflow in the tower was 150 m³/h. The moisture content inthe powder was less than 4% and the powder was characterized by veryhigh flow ability.

The powders of examples 13 and 14, when reconstituted in water, compriseessentially micelles having the same structure and morphology as thewhey protein micelle concentrate.

Example 15 Whey Protein Micelle Powder Obtained by Freeze-Drying

Material

Whey protein micelle concentrate at 20% protein produced bymicrofiltration in example 12 with a protein content of 90%

Method

100 g of whey protein micelles concentrate were introduced in a plasticbeaker and frozen at −25° C. for one week. This beaker was then placedin a lab-scale freeze drier Virtis equipped with a vacuum pump. Samplewas left for 7 days until the pressure in the freeze drier remainedconstant at about 30 mbars. Around 20 g of freeze-dried whey proteinmicelles has been recovered.

Example 16 A Whey Protein Enriched Dark Chocolate without Sucrose

Material Ingredients Percentage Whey protein micelle powder 40-50% fromexample 13 with a protein content of 90% Sucralose 0.05-0.1% Anhydrousmilk fat 3-5% Cocoa liquor 30-40% Cocoa butter 5-15% Vanillin0.005-0.015% Lecithin 0.1-1%Method

Cocoa liquor is mixed with cocoa butter, butter fat, whey proteinmicelle powder, sucralose, vanillin and lecithin. This mixture isconched overnight at 65° C. until a homogenous paste is obtained. Thischocolate mass is then moulded in chocolate plates and cooled down. Thedark chocolate is characterized by a final whey protein content of45-50%.

Example 17 A Whey Protein Enriched White Chocolate

Material Ingredients Method 1 Method 2 Method 3 Whey protein micelle15-25% 25-35% 35-40% powder from example 13 with a protein content of90% Sucrose 40-45% 30-35% 30-35% Anhydrous milk fat  1-10%  1-10%  1-10%Whey powder  2-10%  2-10%   0% Cocoa butter 20-30% 20-30% 20-30%Vanillin 0.01-0.1%  0.01-0.1%  0.01-0.1%  Lecithin  0.1-1%  0.1-1% 0.1-1%Method 1

Whey protein micelles, whey powder, sucrose and vanillin are mixed andground until the desired particle size distribution is obtained. Thismixture is then conched overnight at 65° C. with cocoa butter, anhydrousmilk fat and lecithin until a homogenous paste is obtained. Thischocolate mass is then moulded in chocolate plates and cooled down. Thiswhite chocolate is characterized by a final whey protein content of 20%.

Method 2

Whey protein micelles, whey powder, sucrose and vanillin are mixed andground until the desired particle size distribution is obtained. Thismixture is then conched overnight at 65° C. with cocoa butter, anhydrousmilk fat and lecithin until a homogenous paste is obtained. Thischocolate mass is then moulded in chocolate plates and cooled down. Thiswhite chocolate is characterized by a final whey protein content of 30%.

Method 3

Whey protein micelles, sucrose and vanillin are mixed and ground untilthe desired particle size distribution is obtained. This mixture is thenconched overnight at 65° C. with cocoa butter, anhydrous milk fat andlecithin until a homogenous paste is obtained. This chocolate mass isthen moulded in chocolate plates and cooled down. This white chocolateis characterized by a final whey protein content of 30-35%.

Example 18 Aqueous Dispersion of Whey Protein Micelles Coated withSulfated Butyl Oleate (SBO) or any Other Negatively Charged Emulsifier

Material

Whey protein micelle (WPM) powder from example 13 with a protein contentof 90%

SBO

Hydrochloric acid (1M)

Method

WPM powder described in example 13 is dispersed in MilliQ water toachieve a final protein concentration of 0.1 wt %. This dispersion isfiltered on 0.45 μm filters in order to remove possible WPM aggregates.The pH of this WPM dispersion was brought down to 3.0 by addition ofhydrochloric acid 1M. A 1 wt % dispersion of SBO is prepared at pH 3.0.

The hydrodynamic radius and zeta potential of these WPM was determinedusing the Nanosizer ZS apparatus (Malvern Instruments Ltd.). Diameterwas 250 nm and electrophoretic mobility+2.5 μm.cm.V⁻¹.s⁻¹. Thehydrodynamic radius and electrophoretic mobility of the SBO dispersionat pH 3.0 are 4 nm and −1.5/−2.0 μm.cm.V⁻¹.s⁻¹, respectively.

After having performed this preliminary characterization, the SBOdispersion is used to titrate the WPM one, while following evolution ofhydrodynamic radius and electrophoretic mobility of the mixture. It wasfound that the hydrodynamic radius was constant around 250-300 nm untila WPM/SBO weight-mixing ratio of 5:1 was reached. At this point, thehydrodynamic radius diverges dramatically to 20000 nm and precipitationof complexes WPM SBO is encountered. Upon further addition of SBO,higher than a mixing ratio of 5:1, the hydrodynamic progressivelydecreased to 250 nm, as found initially for WPM, levelling of from aratio of 4:1 on. Following the electrophoretic mobility of the mixtureshowed that it decreased upon addition of SBO, reaching zero value for amixing ratio of 5:1. Then it continued to drop upon SBO addition,starting levelling of at −3.0 μm.cm.V⁻¹.s⁻¹ from ratio 4:1 on.

The explanation for these results is that the positively charged WPMare, in a first step coated electrostatically with the negative head ofthe SBO until full charge neutralisation is achieved (mixing ratio 5:1).At this point, the hydrophobic tails from the SBO are able toself-associate, leading to over-aggregation with very large hydrodynamicdiameter and precipitation of complexes. Upon further addition of SBO,the hydrophobic tails associate further to form a double coating,exposing their negative head to the solvent. This lead to negativelycharged WPM with a double coating of SBO (see FIG. 17) comparable to afull protein core liposome.

Similar results have been obtained with other acidic food gradeEmulsifiers such as DATEM, CITREM, SSL (from Danisco) in aqueoussolution at pH 4.2 where they are mainly ionized in their anionic form(—COO⁻ chemical functions).

Example 19 A Protein-Enriched Béchamel Sauce

Material

Mixed whey protein micelle powder from example 14 with a protein contentof 70%

Butter

Flour

Skim milk

Salt

Method

30 g of mixed whey protein micelle powder are dispersed in 1 liter ofskim milk under heating. 30 g of butter and 80 g of flour are then addedtogether with 2.85 g of salt. The mixture is then boiled in order toproduce a béchamel sauce having a whey protein content of about 3 g/100g.

Example 20 A Whey Protein-Enriched Base for Performance Bar

Material Ingredients Percentage Mixed whey protein micelle powder 40-50%from example 13 with a protein content of 90% (moisture 3.5%) Brown ricesyrup 35-45% Maltitol  5-10% Glycerol 10-15%Method

Brown rice syrup is mixed with maltitol and glycerol at 25° C. Wheyprotein micelle powder is then added and mixing is performed for 10minutes. A whey protein-enriched base for performance bar is thenobtained and can be mixed with other ingredients (minerals, vitamins,flavours). This preparation contains more proteins than milk (38%).

Example 21 Determination of Repose Angle for Spray Dried Whey ProteinMicelle Powder, Mixed Whey Protein Micelle Powder, Whey Protein IsolatePowder and Low Heat Skim Milk Powder

Material

Whey protein micelle powder from example 12 with a protein content of90% (moisture 3.5%)

Mixed whey protein micelle powder from example 13 with a protein contentof 90% (moisture 3.5%)

Whey protein isolate powder Prolacta 90 (lot 500658 from Lactalis,France; moisture 4%)

Low heat skim milk powder (lot 334314 from Emmi, Switzerland; moisture3.5%)

Measuring device described to measure repose angle for powders accordingto ISO norm 4324

Method

The powder is placed in a funnel with a stem diameter of 99 mm and thepowder is forced to flow using the agitator. The powder falls on atransparent plastic vessel with diameter 100 mm and a height of 25 mm.The angle of repose, Φ, is measured from the following equation:Repose angle Φ=ARCTAN(2h/100)

Where h is the maximum height of the powder cone than can be obtained,all surface of the plastic vessel being covered with powder.

Results from the repose angle test (values are mean of 3 measurementsand standard deviation is indicated).

Whey Mixed whey protein protein Whey Low heat micelle micelle proteinskim milk powder powder isolate powder Repose angle (°) 24.6 ± 1.1 27.3± 0.7 34.3 ± 0.5 43.8 ± 2.8

Repose angle results clearly show that whey protein micelle powder, pureor mixed with maltodextrins, exhibit a significantly lower angle thanthe initial whey protein powder or even skim milk powder. A repose anglelower than 35° is characteristic of very well flowing powders.

1. A process for the production of a whey protein micelles concentrateconsisting of the steps of: a. adjusting the pH of a demineralized wheyprotein aqueous solution to a value between 5.8 and 6.6 wherein theconcentration of the whey protein aqueous solution is less than 12%,wherein if the whey protein aqueous solution comprises additionalcompounds, the amount of the additional compounds does not exceed 10% byweight of the total weight of the solution; b. subjecting the aqueoussolution to a temperature of between 80 and 89 C, wherein the heating iscarried out for a time range of 10 s to 2 hours; and c. concentrating adispersion obtained in step b; wherein in the concentrated dispersion,the proportion of micelles with an average size of between 100 nm and700 nm is greater than 80%.
 2. The process of claim 1, wherein the wheyprotein aqueous solution concentration is less than 4%.
 3. The processof claim 1, wherein the aqueous solution is heated for a time of 15minutes.
 4. The process according to claim 1, wherein the heating isperformed by microwaves.
 5. The process according to claim 1, whereinthe yield of micelles before concentration is at least 35%.
 6. Theprocess according to claim 5, wherein the yield of micelles beforeconcentration is at least 50%.
 7. The process according to claim 5,wherein the yield of micelles before concentration is at least 80%. 8.The process according to claim 1, wherein the concentration is performedby a method selected from the group consisting of evaporation,centrifugation, sedimentation, ultrafiltration and microfiltration. 9.The process of claim 8, wherein the centrifugation is performed afteracidification to a pH of 4.5.
 10. The process of claim 8, whereinspontaneous sedimentation is performed at a pH of 4.5.
 11. The processof claim 10, wherein the sedimentation time is greater than 12 hours.12. The process according to claim 1, wherein the concentrating stepincludes spray-drying or freeze-drying the whey protein micellesconcentrate.
 13. The process of claim 12, wherein the spray-drying orfreeze-drying is performed with additional ingredients.
 14. The processof claim 13, wherein the additional ingredients are selected from thegroup consisting of soluble or non-soluble salts, peptides, proteinhydrolysates, probiotic bacteria, strains, sugars, maltodextrins, fats,emulsifiers, sweeteners, aroma, plant extracts, ligands, bioactiveagents, caffeine, vitamins, minerals, drugs, milk, milk proteins,skimmed milk powder, micellar casein, caseinate, vegetal protein, aminoacids, polyphenols, pigment and mixtures thereof.
 15. A method forpreparing a product selected from the group consisting of nutritional,cosmetic, and pharmaceutical applications consisting of the steps of: a.adjusting the pH of a demineralized whey protein aqueous solution to avalue between 5.8 and 6.6, wherein the concentration of the whey proteinaqueous solution is less than 12%, wherein if the whey protein aqueoussolution comprises additional compounds, the amount of the additionalcompounds does not exceed 10% by weight of the total weight of thesolution; b. subjecting the aqueous solution to a temperature of between80 and 89° C., wherein the heating is carried out for a time range of 10s to 2 hours; and c. concentrating a dispersion obtained in step b andusing the concentrate to prepare the product.